Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.

Abstract

The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP(+)EGFR(+)CD24(-)), niche astrocytes (GFP(+)EGFR(-)CD24(-)), transit amplifying cells (GFP(-)EGFR(+)CD24(-)), and neuroblasts (GFP(-)EGFR(-)CD24(low)). One in three EGFR(+) astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphere-forming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.

Prospective isolation of stem cells and their progeny from the adult SVZ. (A) Schema of a sagittal section of the adult mouse brain showing the lateral ventricle/SVZ (purple) and the SVZ stem cell lineage. Other SVZ astrocytes and ependymal cells lining the ventricles are also present in this region. Table shows the expression of markers used for the prospective isolation of each SVZ cell type. (B) FACS plots of the purification of each SVZ cell type from adult GFAP::GFP transgenic mice. GFAP-positive SVZ astrocytes were separated based on GFP expression from other GFP− SVZ cells. (B′) From the GFP+ pool, we isolated activated stem cell astrocytes (GFP+EGFR+CD24−) from other SVZ astrocytes (GFP+EGFR−CD24−) based on EGFR expression. The GFP+EGFR+ cell fraction largely corresponds to dim-GFP-expressing cells. (B″) From the GFP-negative cell fraction, we isolated transit amplifying cells (GFP−EGFR+CD24−) and neuroblasts (GFP−EGFR−CD24low). Percentages represented by each population are shown next to the gates and are averages of 10 independent experiments. (C) A DNA dye was used to separate the cells according to their cell-cycle status (G0/G1 phases from S/G2–M phases). Percentages represented by each population are shown next to the gates. See Fig. S2 for an example of a plot using Vybrant DyeCycle. All FACS data are presented using “logical” or biexponential display for 100,000 cells. Gates were determined by using control samples shown in Fig. S2.

Purity and marker expression of FACS-purified SVZ populations. (A) Schema represents the SVZ stem cell lineage and the markers enriched at each cell stage. Activated stem cell astrocytes give rise to rapidly dividing transit amplifying cells that differentiate into migrating neuroblasts and eventually give rise to mature neurons of the olfactory bulb. A small percentage of SVZ stem cells differentiate into oligodendrocytes (not shown in the schema). Note that some markers are not specific to one cell type and persist into the following stage of the lineage. (B) Acute immunostaining of SVZ populations purified by FACS from the adult mouse brain. FACS-purified SVZ astrocytes (GFP+), activated stem cell astrocytes (GFP+EGFR+), transit amplifying cells (EGFR+), and neuroblasts (CD24low) were stained with antibodies against GFAP, GLAST, Mash1, and βIII-tubulin (TuJ1) to analyze the purity of each fraction. (Scale bar, 20 μm.) (C) Quantification of acute immunostaining. Data represent the mean percentage ± SEM of cells expressing the different markers in each of the FACS-sorted populations from 5 independent experiments. (D) Relative quantities of gfap, mash1, and βIII tubulin mRNA detected by qRT-PCR in each of the FACS-sorted populations. Data represent relative quantity normalized to gapdh. (E) Quantification of acute double immunostaining for GFAP/Mash1 (Left) and GLAST/Mash1 (Right) in the activated stem cell population (GFP+EGFR+) and transit amplifying (EGFR+) populations. Images of the acute immunostainings are shown in Fig. S4.